Glaucoma is a major cause of blindness affecting over 67 million persons worldwide. Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma and reducing IOP is the only current effective treatment for all forms of glaucoma. Normally, cells within the juxtacanalicular region (JCT) of the trabecular meshwork (TM) and/or Schlemm's canal (SC) inner wall endothelium, sustain IOP homeostasis by adjusting the outflow resistance, thought to reside within this region. In the last funding cycle, we identified a likely component of the outflow resistance, which changes in response to manipulations that modulate the outflow resistance, i.e. mechanical stretch, TNF?, IL-1? and TGF?. Versican, a large chondroitin sulfate proteoglycan, supported by hyaluronan, a large glycosaminoglycan (GAG), may be an important component of the outflow resistance. These studies accentuated the highly segmental nature of outflow around the circumference of the eye. Although flow segmentation increases the complexity of outflow pathway analysis, it also provides an ideal venue to study the outflow resistance. The focus of this current proposal is to develop a more complete understanding of the molecular structure and organization of outflow resistance. The general approach will be to compare properties of high and low flow regions in normal and glaucomatous eyes both before and after they have undergone the resistance adjustment produced by the IOP homeostatic response. In the first specific aim we will: A) develop a detailed map of segmental flow patterns; B) determine whether the IOP homeostatic flow resistance change occurs in high or in low flow regions; and C) compare the biomechanical compliance of the JCT in high vs. low flow regions. In the second specific aim we will compare molecular ECM composition between high and low flow regions: A) at the mRNA level and isoform usage; B) at the protein level using ECM protein enrichment and proteomics; and C) localization and distribution by confocal immunohistochemistry and binding protein patterns. In the third specific aim we will test the involvement of versican and other components identified in aims 2 and 3 in the organization and regulation of the outflow resistance using: A) selective versican isoform RNAi silencing; B) selective perturbations of ECM components; and C) binding partner analysis. These studies will enhance our understanding of the molecular organization and regulation of the outflow resistance and should provide new potential therapeutic targets for glaucoma.